The invention relates essentially to the use of collagen of aquatic origin for the production of supports for tissue engineering, and to such supports and biomaterials.
Collagen is a particularly favorable substrate for cell development, which is why this protein is very widely used in several formsxe2x80x94matrices, gels or filmsxe2x80x94for the production of reconstructed tissues containing living cells.
In the field of tissue engineering, a technique which promises to have a great future, collagen has afforded the production of artificial skin or cartilage in particular. To achieve a satisfactory result, the collagen has to be protected from enzymatic degradation due to cell metabolism, either by physical or chemical crosslinking processes, or by the presence of natural macromolecules which interact strongly with the protein, or finally by a combination of both systems.
Hitherto, for these tissue engineering applications, the collagen used in supports for receiving cells was extracted from mammals and most frequently from bovine skin. The choice of this source was due to the good mechanical properties of the protein obtained after extraction, to its resistance to enzymatic degradation and finally to its amino acid composition, which is very similar to that of human collagen. For all these reasons, it was legitimate to think that this collagen was the only one suitable for the culture of human cells.
Now, the inventors have noticed, unexpectedly, that human cells develop very well on or inside certain supports consisting of preferably crosslinked fish collagen. In addition, the inventors have been able to demonstrate that human cells cultivated in these biomaterials preserve a normal metabolism. These biomaterials can be either films, or compressed sponges, or porous matrices, which will be described together with their methods of preparation in the Examples given below.
One object of the present invention is to solve the new technical problem which consists in providing novel supports for tissue engineering suitable for forming novel biomaterials, i.e. suitable for allowing a good proliferation of the normal, genetically modified or malignant living cells to be cultivated on said support and to be used within the framework of these novel biomaterials, containing said living cells, for subsequent proliferation in vitro or in vivo.
A further object of the present invention is to solve the new technical problem which consists in providing novel supports for tissue engineering at a low manufacturing cost and also with a low risk of contamination, thus making them particularly suitable for the provision of novel biomaterials.
A further main object of the present invention is to solve the new technical problem which consists in providing novel supports for tissue engineering which are particularly suitable for allowing the multiplication of normal, genetically modified or malignant living cells, in vitro or in vivo, and whose structure is sufficiently compatible with in vivo use in a mammal, particularly an animal or, preferably, a human being, while at the same time being different from the constitution of the tissues of said mammal, such as an animal or, preferably, a human being, so as to allow subsequent differentiation between the newly synthesized tissues and the old tissues of said mammal, preferably a human being.
The present invention solves all these technical problems for the first time in a satisfactory manner, at low cost, with a low risk of contamination or without contamination, while at the same time easily making it possible to identify the newly synthesized tissues, which is particularly non-obvious and unexpected for those skilled in the art.
Thus, according to a first feature, the present invention relates to the use of collagen of aquatic origin for the production of supports for tissue engineering, as well as the corresponding method of production thereof.
The expression xe2x80x9ccollagen of aquatic originxe2x80x9d is understood as meaning a collagen derived from collagen-containing tissues of living beings of aquatic origin; these living beings are well known to those skilled in the art and include for example, without implying a limitation, aquatic mammals, particularly marine mammals, jellyfish and saltwater or freshwater fish. Furthermore, those skilled in the art know that the skin of these living beings contains essentially collagen.
In one advantageous embodiment, the collagen is obtained from fish skin, preferably in its native form.
In another advantageous embodiment of the invention, the mechanical strength of the collagen or its resistance to enzymatic digestion is increased either by chemical and/or physical crosslinking, or by the addition of a natural macro-molecule which interacts strongly with collagen, or by a combination of both processes.
In yet another advantageous embodiment of the invention, the collagen is used in the form of a porous matrix prepared from a collagen gel which has preferably undergone a lyophilization step.
In yet another advantageous variant, the above-mentioned porous matrix is crosslinked by a physical method, preferably by thermal dehydration, or TDH.
In yet another advantageous variant, the above-mentioned porous matrix is crosslinked by a chemical method, preferably with diphenylphosphorylazide, or DPPA, or with a carbodiimide and/or N-hydroxysuccinimide, or with glutaraldehyde.
In one advantageous embodiment, the above-mentioned collagen can take the form of a porous matrix prepared from marine collagen (preferably native) mixed with chitosan and optionally at least one glycosaminoglycan, preferably chondroitin sulfate.
In yet another advantageous embodiment of the invention, the above-mentioned collagen can take the form of a porous matrix prepared from a collagen gel, said porous matrix being covered on at least one side with an essentially compact collagen membrane consisting either of a collagen film prepared by drying a collagen gel, preferably in air or a gaseous fluid, or of a very highly compressed collagen sponge.
In another advantageous variant, the above-mentioned compression of the very highly compressed collagen sponge is carried out at a pressure of at least about 50 bar (about 50.105 Pascal (Pa)) and preferably of between 50 bar (50.105 Pa) and 200 bar (200.105 Pa), this compression optionally having been carried out at a temperature of between 20xc2x0 C. and 80xc2x0 C. and preferably of between 40xc2x0 C. and 60xc2x0 C.
According to yet another advantageous characteristic of the invention, at least one of the two layers, i.e. the porous layer and the essentially compact membrane, comprises normal, genetically modified or malignant living cells originating particularly from young or elderly subjects.
In one advantageous embodiment, the living cells are selected from the group consisting of fibroblasts, keratinocytes, melanocytes, Langerhans"" cells originating from the blood, endothelial cells originating from the blood, blood cells, particularly macrophages or lymphocytes, adipocytes, sebocytes, chondrocytes, osteocytes, osteoblasts and Merkel""s cells originating from the blood, said cells being normal, genetically modified or malignant.
In one particularly advantageous embodiment, the porous layer contains normal, genetically modified or malignant fibroblasts and the essentially compact membrane contains normal, genetically modified or malignant living cells selected particularly from keratinocytes, melanocytes, Merkel""s cells originating from the blood, Langerhans"" cells originating from the blood, sebocytes, cells originating from the blood, and nerve cells.
In yet another advantageous embodiment of the invention, it may be of particular value to prepare either xe2x80x9cyoungxe2x80x9d reconstructed skin using cells taken from young subjects, or xe2x80x9cagedxe2x80x9d reconstructed skin obtained from cells taken from elderly subjects. These models will enable us to improve our knowledge of the skin ageing process and study the influence of active agents on this process.
In another particularly advantageous embodiment, the above-mentioned essentially compact membrane is prepared prior to combination with the porous layer, preferably comprising a collagen sponge, particularly by preparing the membrane and depositing it on a collagen gel before the whole is frozen and lyophilized.
According to a second feature, the present invention also covers a support for tissue engineering which comprises collagen of aquatic origin as defined above or as resulting from the following description taken in its entirety and including the Examples, which form an integral part of the present invention in their generality, and as regards any characteristic which appears to be novel by comparison with any state of the art, this characteristic being taken in its function and in its generality, independently of the context of the Example.
According to a third feature, the present invention also covers a biomaterial, for example in the form of a reconstituted connective tissue or reconstituted skin, which has been prepared from the collagen of aquatic origin as defined above in all these features and also as resulting from the following description, as for the support of the second feature above.
within the framework of the present description and the claims, the expression xe2x80x9csupports for tissue engineeringxe2x80x9d denotes supports to be used for carrying out the culture and proliferation of normal, genetically modified or malignant living cells, whether in vitro or in vivo, this proliferation preferably being applied in vivo to a mammal, comprising an animal and, preferably, a human being. It is understood that the invention has a particularly preferred use within the framework of tissue engineering for the manufacture of biomaterials, for example in the form of reconstituted connective tissues or reconstituted skin. within this framework, a first step will generally be the culture of the support with said living cells in vitro to give a biomaterial, for example in the form of a reconstituted connective tissue or reconstituted skin, and then a second step will be the use of this biomaterial as reconstituted connective tissue or reconstituted skin in vivo on a mammal, for example an animal or, preferably, a human being, in order to reconstitute a connective tissue damaged or removed by surgery or, likewise, in order to reconstitute skin to replace an area damaged or removed by surgery for whatever medical reason.
Advantageously, the support for tissue engineering or, preferably, the biomaterial, for example in the form of a reconstituted connective tissue or reconstituted skin, comprises cells obtained either substantially exclusively from young subjects or substantially exclusively from elderly subjects, in particular for studying the tissue ageing process, and especially the skin ageing process, and optionally for testing the efficacy of active ingredients or principles on this process.
The invention further relates to an artificial skin which is essentially prepared from substantially exclusively young cells originated from young subjects.
The invention further relates to an artificial skin which is essentially prepared from substantially exclusively aged cells originated from elderly subjects.
The invention further relates to an artificial skin comprising living cells essentially prepared from substantially exclusively young cells originated from young subjects.
The invention further relates to an artificial skin comprising living cells essentially prepared from substantially exclusively aged cells originated from elderly subjects.
According to a further aspect, the invention relates to an artificial skin comprising a support comprising or substantially exclusively constituted from marine collagen, preferably fish collagen.
The invention further relates to a method of in vitro testing of the efficacy of a potential active substance comprising using an artificial skin comprising living cells essentially prepared substantially exclusively from young cells taken from young subjects, in combination with a support comprising or essentially consisting of marine collagen.
The invention further relates to a method of in vitro testing of the efficacy of a potential active substance comprising using an artificial skin comprising living cells essentially prepared substantially exclusively from aged cells taken from elderly subjects, in combination with a support comprising or essentially consisting of marine collagen.
The invention further relates to a method of reconstructing damaged areas of skin in vivo comprising performing said reconstruction with an artificial skin comprising living cells, and prepared essentially from a support comprising or essentially consisting of marine collagen, preferably fish collagen.
In each and all of the above features, aspects or embodiments of the invention, it is advantageous that at least one of the porous matrix or layer, and of the essentially compact membrane is produced from a collagen gel containing a mixture of soluble collagen and insoluble collagen. Preferably, said insoluble collagen is comprising or is substantially essentially consisting of collagen fibers.
According to another advantageous feature, at least a part of, or substantially all of, the collagen is selected from the group consisting of type I collagen and type III collagen, said feature being of course combinable with any other feature of the present invention.
Thus it is seen that the invention provides a general solution to the above-mentioned new technical problems in a particularly simple manner, at low cost, with a low risk of contamination and with a capacity to differentiate between aquatic collagen and mammalian collagen or, preferably, human collagen, newly synthesized in the course of in vivo use.
In fact, the use of fish collagen in the production of living artificial tissues has three essential advantages compared with the mammalian source:
The fish skin generally used as the raw material can be obtained in abundance under very clean conditions.
The danger of infectious contamination is very low. In particular, there is no known risk of transmitting agents of the prion type.
Finally, as the amino acid composition of fish collagen is relatively dissimilar to that of human collagen, the two proteins can be differentiated relatively easily by means of specific antibodies. This methodology will be very valuable particularly in xe2x80x9cin vitroxe2x80x9d tests or in xe2x80x9cin vivoxe2x80x9d healing studies.
Moreover, the use of marine collagen will make immunolabeling very effective and allow differentiation between marine collagen and newly synthesized collagen.
Fish collagen has a native structure which protects it from enzymatic degradation due to proteases and which is largely responsible for its mechanical properties. It will therefore be very important that the treatments used during the extraction and purification operations degrade the protein structure as little as possible. This means that the helical structure and the intermolecular and intramolecular crosslinks should be preserved as far as possible. The inventors achieved this more particularly by implementing the process described in patent U.S. Pat. No. 5,331,092 granted on Jul. 19, 1994. Nevertheless, for particular applications, it may be possible to envisage the use of partially decrosslinked collagen, for example atelocollagen, i.e. collagen which has lost part of its telopeptides.
Then, for the majority of tissue engineering applications, the mechanical properties of the collagen will be enhanced and its resistance to enzymatic digestion increased either by chemical and/or physical crosslinking techniques, or by the addition of natural macromolecules which interact strongly with the protein, or finally by a combination of both processes.
The protection of fish collagen will be all the more important because its natural stability is lower than that of mammalian collagen, the latter characteristic being due to a lower hydroxyproline content.
The biomaterials described above may be inoculated with living cells to create living artificial tissues which may be used either in the field of xe2x80x9cin vitroxe2x80x9d tests or in the pharmaceutical field for repairing injured tissues.
Other objects, characteristics and advantages of the invention will become clearly apparent from the following explanatory description referring to Examples of the preparation of forms of collagen of aquatic origin which can be used within the framework of the invention for the production of supports for tissue engineering, and thus constituting such supports as well as biomaterials, which are given simply by way of illustration and cannot therefore in any way limit the scope of the invention.
In the Examples, the temperature is given in degrees Celsius, the pressure is atmospheric pressure and the percentages are given by weight, unless indicated otherwise.
Examples 1 to 13 are of course Examples of the preparation of collagen which can be used as a tissue engineering support according to the invention.
Examples 14 to 16 are notably comparative tests within the framework of the use of this collagen of aquatic origin, in the forms prepared in some of Examples 1 to 13, within the framework of the production of supports for tissue engineering.